Assay and method for predicting therapeutic efficacy of immunoglobulin therapy in individual patients with relapsing remitting multiple sclerosis (rr-ms)

Abstract

A method for generating a reference for determining the likelihood of response of a patient, suffering from a disease, towards immunoglobulin therapy comprising the steps of a) providing samples of a sufficient number of individuals, in particular at least 10 individuals, the samples containing B- and T-lymphocytes, natural killer cells, invariant T-cells and monocytes of the individuals; b) determination of values of immune parameters which are either static, such as leukocyte subpopulations and cytokine-level in the plasma or functional, like gene expression and cytokine release after lipopolysaccharide (LPS) and/or IVIG stimulation ex vivo; c) the determined values derived from immune parameters of the samples of the individuals are ordered in quartiles and the values belonging to the 1. quartile, values distributed at the low end of the corresponding parameter, are set to “−1”, whereas values distributed in the 4. quartile are set to “+1” for an individual LDA-score calculation and values in between are set to “0”; d) genotyping of at least two of the polynucleotides selected from the group consisting of MTM1, EIF3E, COPS8, ADAMTSL1, CXXC4, RSPO2, OR9Q1, ADAMTS9, KLHDC8A and PRDM9, in the samples of the individuals and awarding the value of 0 for specific homozygous SNP combinations (SNP—Single Nucleotide Polymorphism), which indicates that the blood sample stems from a person which will respond to immunoglobulin (IG) treatment, while awarding the value of 1 for SNP combinations not meeting that criteria, which indicates that the blood sample stems from a person which will not respond to immunoglobulin treatment; e) combining the results of genotyping with results of immuno parameter determination in the calculation of an individual LDA-score (LDA—Linar Discriminant Analysis); f) combining a multiplicity of individual LDA-scores to create a reference Responder Score, which allows discrimination between responders and non-responders. The method can be employed for determining the likelihood of response of a patient, suffering from a disease, towards immunoglobulin therapy.

1 . A method for generating a reference for determining the likelihood of response of a patient, suffering from a disease, towards immunoglobulin therapy comprising the steps of
 a) providing samples of a sufficient number of individuals, the samples containing B- and T-lymphocytes, natural killer cells, invariant T-cells or monocytes of the individuals;   b) determination of values of immune parameters which are either static or functional   c) the determined values derived from immune parameters of the samples of the individuals are ordered in quartiles and the values belonging to the 1. quartile, values distributed at the low end of the corresponding parameter, are set to “4”, whereas values distributed in the 4. quartile are set to “+1” for an individual LDA-score calculation and values in between are set to “0”;   d) genotyping of at least two polynucleotides selected from the group consisting of MTM1, EIF3E, COPS8, ADAMTSL1, CXXC4, RSPO2, OR9Q1, ADAMTS9, KLHDC8A and PRDM9, in the samples of the individuals and awarding the value of 0 for specific homozygous SNP combinations (SNP—Single Nucleotide Polymorphism), which indicates that the blood sample stems from a person which will respond to immunoglobulin (IG) treatment, while awarding the value of 1 for SNP combinations not meeting that criteria, which indicates that the blood sample stems from a person which will not respond to immunoglobulin treatment;   e) combining the results of genotyping with results of immuno parameter determination in the calculation of an individual LDA-score (LDA—Linar Discriminant Analysis);   f) combining a multiplicity of individual LDA-scores to create a reference Responder Score, which allows discrimination between responders and non-responders.   
     
     
         2 . The method of  claim 1  wherein the immune parameters are selected from the group consisting of CD45, CD3, CD56, CD16, CD19, CD 14, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL13, ICAM1, sICAM1 (soluble ICAM1), IL-1RA, CCL2, IFN-γ, Actin-β, and PPIB. 
     
     
         3 . The method of  claim 1  wherein the homozygous SNP combination related to the KLHDC8A gene is represented by dbSNP RS ID's rs7549293-rs10751436-rs913723-rs913722 with the SNP combination CC-TT-AA-TT (in the same order as given dbSNP RS ID's) indicating a non-responder and contributing “1” for individual LDA-score calculation, while all different SNP combinations indicate a responder and contribute “0” for the individual LDA-score calculation. 
     
     
         4 . The method of  claim 1  wherein the homozygous SNP combination related to the PRDM9 and OR9Q1 genes is represented by dbSNP RS ID's rs13182871-rs7701403-rs12576939-rs1376486 with the SNP combination GG-AA-TT-GG (in the same order as given dbSNP RS ID's) indicating a non-responder and contributing “1” for individual LDA-score calculation, while all different SNP combinations indicate a responder and contribute “0” for the individual LDA-score calculation. 
     
     
         5 . The method of  claim 1  wherein the homozygous SNP combination related to the ADAMTS9 gene is represented by dbSNP RS ID's rs9820942-rs6780659-rs6445415-rs11721258-rs11707584-rs7652817-rs13079218-rs9819183 with the SNP combination GG CC AA TT AA GG TT AA (in the same order as given dbSNP RS ID's) indicating a non-responder and contributing “1” for individual LDA-score calculation, while all different SNP combinations indicate a responder and contribute “0” for the individual LDA-score calculation. 
     
     
         6 . The method of  claim 1  wherein the homozygous SNP combination related to the MTM1 gene is represented by dbSNP RS ID's rs222382-rs222397-rs222396-rs222385 with the SNP combination GG-AA-AA-TT (in the same order as given dbSNP RS ID's) indicating a responder and contributing “0” for individual LDA-score calculation, while all different SNP combinations indicate a non-responder and contribute “1” for the individual LDA-score calculation. 
     
     
         7 . The method of  claim 1  wherein the homozygous SNP combination related to the EIF3E gene is represented by dbSNP RS ID's rs617285-rs596747-rs16877589 with the SNP combination TT-AA-TT (in the same order as given dbSNP RS ID's) indicating a responder and contributing “0” for individual LDA-score calculation, while all different SNP combinations indicate a non-responder and contribute “1” for the individual LDA-score calculation. 
     
     
         8 . The method of  claim 1  wherein the homozygous SNP combination related to the COPS8 gene is represented by dbSNP RS ID's rs1391108-rs10194251-rs12477269-rs6732507 with the SNP combination AA-TT-CC-AA (in the same order as given dbSNP RS ID's) indicating a responder and contributing “0” for individual LDA-score calculation, while all different SNP combinations indicate a non-responder and contribute “1” for the individual LDA-score calculation. 
     
     
         9 . The method of  claim 1  wherein the homozygous SNP combination related to the ADAMTSL1 gene is represented by dbSNP RS ID's rs507741-rs550436-rs577994-rs560843-rs506193 with the SNP combination GG-GG-TT-AA-CC (in the same order as given dbSNP RS ID's) indicating a responder and contributing “0” for individual LDA-score calculation, while all different SNP combinations indicate a non-responder and contribute “1” for the individual LDA-score calculation. 
     
     
         10 . The method of  claim 1  wherein the homozygous SNP combination related to the CXXC4 gene is represented by dbSNP RS ID's rs3017683-rs886332-rs2214397-rs2189216 with the SNP combination TT-GG-TT-GG (in the same order as given dbSNP RS ID's) indicating a non-responder and contributing “1” for individual LDA-score calculation, while all different SNP combinations indicate a responder and contribute “0” for the individual LDA-score calculation. 
     
     
         11 . The method of  claim 1  wherein the homozygous SNP combination related to the RSPO2 gene is represented by dbSNP RS ID's rs446454-rs388681-rs370662-rs399085-rs442355-rs377057-rs425932-rs564791-rs404930-rs375444-rs609666 with the SNP combination GG AA TT CC GG GG AA GG CC-CC-CC (in the same order as given dbSNP RS ID's) indicating a responder and contributing “0” for individual LDA-score calculation, while all different SNP combinations indicate a non-responder and contribute “1” for the individual LDA-score calculation. 
     
     
         12 . The method of  claim 1  wherein the multiplicity of immune parameters used for the creation of individual LDA-scores is 4 at maximum. 
     
     
         13 . The method of  claim 1  wherein the genotyping result of the polynucleotides related to the genes of ADAMTS9, KLHDC8A and PRDM9 are not complemented for creation of individual LDA-scores and represent their own individual LDA-score. 
     
     
         14 . The method of  claim 1  wherein the individual LDA-score related to the MTM1 genotype is calculated by the formula:
   Individual  MTM 1-LDA-score=4.664*( MTM 1 genotype result)+1.553*(IG induced(netto) CD 16 gene expression)−1.063*( CXCL 2 plasma concentration)−2.168*(LPS induced  CCL 2 gene expression ratio)−2.185,
 
 
       wherein values lower than the maximal Youden Index indicate responders and are awarded a “0” for the Responder Score calculation, while values above indicate non-responders and are awarded a “1”. 
     
     
         15 . The method of  claim 1  wherein the individual LDA-score related to the EIF3E genotype is calculated by the formula:
   Individual  EIF 3 E -LDA-score=−5.133*( EIF 3 E  genotype result)+1.434*(IG induced(netto) CCL 2 gene expression)+1.988*(IG/LPS induced  CCL 2 gene expression ratio)−0.684*(Proportion of  T -cells)+1.901,
 
 
       wherein values higher than the maximal Youden Index indicate responders and are awarded a “0” for the Responder Score calculation, while values below indicate non-responders and are awarded a “1”. 
     
     
         16 . The method of  claim 1  wherein the individual LDA-score related to the COPS8 genotype is calculated by the formula:
   Individual  COPS 8-LDA-score=−6.442*( COPS 8 genotype result)+1.592*(IG/LPS induced  CXCL 8 gene expression)+0.911*(IG/LPS induced(netto) CCL 2 gene expression)+1.930*(IG/LPS induced(netto) IL -1β gene expression)+3.245,
 
 
       wherein values higher than the maximal Youden Index indicate responders and are awarded a “0” for the Responder Score calculation, while values below indicate non-responders and are awarded a “1”. 
     
     
         17 . The method of  claim 1  wherein the individual LDA-score related to the ADAMTSL1 genotype is calculated by the formula:
   Individual  ADAMTSL 1-LDA-score=3.369*( ADAMTSL 1 genotype result)−1.897*(LPS induced  CXCL 2 gene expression)+1.212*(LPS induced  CXCL 10 gene expression)+1.152*(LPS stimulated  IL -1β gene expression)+0.859*(IG/LPS induced(netto) CXCL 2 gene expression)−1.171,
 
 
       wherein values lower than the maximal Youden Index indicate responders and are awarded a “0” for the Responder Score calculation, while values above indicate non-responders and are awarded a “1”. 
     
     
         18 . The method of  claim 1  wherein the individual LDA-score related to the CXXC4 genotype is calculated by the formula:
   Individual  CXXC 4-LDA-score=2.925*( CXXC 4 genotype result)+1.992*(IG/LPS induced (netto) IFN -γ gene expression)−1.099*(IG induced(netto) CXCL 9 protein release)−1.476,
 
 
       wherein values lower than the maximal Youden Index indicate responders and are awarded a “0” for the Responder Score calculation, while values above indicate non-responders and are awarded a “1”. 
     
     
         19 . The method of  claim 1  wherein the individual LDA-score related to the RSPO2 genotype is calculated by the formula:
   Individual  RSPO 2-LDA-score=2.975*( RSPO 2 genotype result)+0.7941*( sICAM 1 plasma concentration)−1.491,
 
 
       wherein values lower than the maximal Youden Index indicate responders and are awarded a “0” for the Responder Score calculation, while values above indicate non-responders and are awarded a “1”. 
     
     
         20 . The method of  claim 1  wherein 2 to 9 results of individual LDA-scores are combined, by addition, to create the Responder Score and wherein the cut-off to discriminate between responders and non-responders is set by adding 4 times the standard deviation of the reference data to the highest obtained score in the reference responder group. 
     
     
         21 . The method of  claim 20  wherein 9 results of individual LDA-scores are combined, by addition, to create the Responder Score. 
     
     
         22 . The method of  claim 1  wherein the immune parameters are determined after ex vivo exposure of samples with immunoglobulin. 
     
     
         23 . The method of  claim 1  wherein genotyping and immune parameters are determined in whole blood, blood fractions, cell fractions or plasma. 
     
     
         24 . The method according to  claim 1  wherein the amount of immunoglobulins used in assays is from about 0.01 to about 100 mg/ml. 
     
     
         25 . The method of  claim 1  wherein the method is performed before and/or during the treatment of a patient with immunoglobulin. 
     
     
         26 . The method of  claim 1  wherein any immunoglobulin product suitable for in vivo use is concerned. 
     
     
         27 . A method for determining the likelihood of response of a patient, suffering from a disease, towards immunoglobulin therapy comprising the steps of
 a) providing a sample of the patient containing B- and T-lymphocytes, natural killer cells, invariant T-cells and monocytes;   b) determination of values of immune parameters which are either static, such as leukocyte subpopulations and cytokine-level in the plasma or functional, like gene expression and cytokine release after lipopolysaccharide (LPS) and/or IVIG stimulation ex vivo;   c) genotyping of at least two of the polynucleotides selected from the group consisting of MTM1, EIF3E, COPSE, ADAMTSL1, CXXC4, RSPO2, OR9Q1, ADAMTS9, KLHDC8A and PRDM9, and awarding the value of 0 for specific homozygous SNP combinations (SNP—Single Nucleotide Polymorphism);   d) combining the results of genotyping of step c) with results of immuno parameter determination of step b) to calculate a the patient's LDA-score (LDA—Linear Discriminant Analysis);   e) combining the patient's LDA-scores by addition to obtain a patient's Responder score;   f) comparing said patient's Responder score with the Responder score of the reference generated by the method according to at least one of the  claim 1 , wherein the cut-off to discriminate between responders and non-responders is set by adding 4 times the standard deviation of the reference data to the highest obtained score in the reference responder group;   g) and a patient's Responder score below this cut-off indicates a responder, whereas a patient's Responder score above this cut-off indicates a non-responder.   
     
     
         28 . The method according to at least one of the  claim 1  wherein the disease is selected from the group consisting of inflammatory mediated immune diseases, autoimmune diseases, allergies, graft-versus-host reactions and prevention of transplant rejection; any kind of multiple sclerosis or any other demyelinating neurological disease; and relapsing-remitting multiple sclerosis. 
     
     
         29 . The method according to  claim 1  to predict the probability of a relapse of a MS patient and/or the rate of progression of the disease in terms of disability and or functioning of the patient as measured by a clinical scale. 
     
     
         30 . The method of  claim 1  which facilitates the approval or recommendation of immunoglobulins by health authorities for the treatment of any kind of multiple sclerosis or any other demyelinating disease or Lupus erythematosus, rheumatoid arthritis or intestinal/bowel diseases such as Crohn's disease, myositis or recurrent abortion. 
     
     
         31 . The method of  claim 1 , wherein
 (a) samples are provided from at least 10 individuals and/or   (b) the static immune parameters are leukocyte subpopulations or cytokine-level in the plasma; and/or the functional immune parameters are gene expression and cytokine release after lipopolysaccharide (LPS) and/or IVIG stimulation ex vivo.   
     
     
         32 . The method of  claim 1 , wherein
 the samples are exposed to the immunoglobulin IgG, IgM, IgA or a combination thereof and/or   the amount of immunoglobulins used in the assays is from about 1 to about 50 mg/ml and/or   the immununoglobulin product is applied intravenously, subcutaneously, intramuscularly, ocularly, intrathecially, orally, topically or inhalably.   
     
     
         33 . The method of  claim 29 , wherein the clinical scale which is measured comprises the expanded disability status scale (EDSS). 
     
     
         34 . The method of  claim 33 , wherein the EDSS is lupus erythematosis, rheumatoid arthritis, or the intestinal/bowel disease Crohn's disease, myositis or recurrent abortion.